Leishmania are important tropical protozoan parasites, causing disease in more than 10 million people worldwide. By understanding the mechanisms used by this parasite to survive and cause disease, it will be possible to design and develop improved chemotherapy and vaccination strategies. Here we focus on the parasite surface, which is covered by a dense glycocalyx consisting primarily of lipophosphoglycan (LPG). This GPI-anchored polyphosphosaccharide, along with structurally related glycoconjugates, play important roles in parasite survival and virulence. By generating LPG mutants and then isolating genes which rescue this defect, we have previously established a powerful genetic system to probe parasite virulence. We will use this methodology to develop a comprehensive understanding of the LPG biosynthetic pathway, and to understand how specific LPG genes and glycoconjugates contribute to disease. These studies will focus on Leishmania major, the agent of cutaneous leishmaniasis, as it offers many experimental advantages including the ability to examine the complete infectious cycle in well defined animal and insect models, and soon the complete genome sequence will be available. Preliminary data from our laboratory suggest that these studies of L. major LPG are applicable to L. donovani as well, the agent of fatal visceral leishmaniasis. Our specific aims are 1) to use LPG mutants in a forward genetic approach to identify the relevant LPG biosynthetic genes; typically these are 'new' genes whose activity has not been studied previously in any organism; 2) to use 'reverse genetic' approaches, in combination with the Leishmania genome project and bioinformatic strategies, to identify candidate genes, and establish their role in LPG biosynthesis; 3) to create null 'knockout' mutants in LPG genes in a fully virulent parasite background, including relevant controls, and then to use these to map out their effects on the synthesis and structure of LPG family glycoconjugates, and their effect on parasite virulence in tests involving infections of mice, macrophages and sand flies; and 4) to dissect the molecular mechanisms by which virulence is compromised by key structural domains of LPG and related glycoconjugates. As one example, we will study a genetic model (phosphoglycan-deficiency) which separates acute virulence from persistence, and explore its utility in vaccine strategies.